1. Field of the Invention
The present invention relates to a method for producing polyploid plants of orchids by physical cutting and plant tissue culture techniques. More particularly, the present invention relates to the method of utilizing plant tissue culture technique to induce endopolyploid cells, contained in a tissue culture material, to develop into polyploid plants, without using chemical agents (e.g. colchicine or other antimicrotubule agents).
2. Description of the Related Art
Today orchids, particularly Phalaenopsis (known as moth orchid), are one of the most important flower products for the markets in the United States of America and also in other countries. In order to maintain the superiority in the competitive market of Phalaenopsis , there is a need of developing new types of Phalaenopsis and improving native species of Phalaenopsis for the diversification of the product.
Most of the wild native Phalaenopsis species are diploids, and the tetraploid cultivars, however, are being successfully developed for the market in recent years. Therefore, the tetraploid Phalaenopsis hybrids have become popular in the market due to the fact that the tetraploid plants have bigger inflorescence and higher quality of their corolla and pedicels. On the other hand, only a few wild species of Phalaenopsis are tetraploid and are used in the breeding program to produce new hybrids or cultivars. Consequently, the genetic base of the cultivars of Phalaenopsis in the market nowadays is considerably inadequate, and the achievement of breeding for the new-type cultivars is limited. To obtain a superior new-type cultivar by integrating the desired characteristics (e.g. fragrance and color) of a wild species of Phalaenopsis , the orchid breeders attempted to develop new triploid cultivars by interspecific hybridization of the wild species of diploid and tetraploid. However, most of the triploid cultivars are highly sterile, and therefore cannot be further improved. Hence, there is a need of providing a simplified and effective breeding technique to obtain new polyploid cultivars of Phalaenopsis . 
Currently, chemical agents are widely used for the chemical induction of chromosome doubling to produce ployploid plants in orchids. Several papers known in the arts demonstrate the usefulness of this approach for the above purpose. For example, the method of Phalaenopsis spp. disclosed by R. J. Griesbach (1981), Plant Cell Tissue Organ Culture, 1:103-107 and R. J. Griesbach (1985), The Journal of Heredity, 76:74-75; the method of Cattleya intermedia disclosed by P. A. K. X. de Mello e Silva et al. (2000), Ciência Rural, Santa Maria, 30:105-111; the method of Cymbidium Silky disclosed by M. S. Kim et al. (2003), Proceedings of NIOC2003 Nagoya, Japan, pp. 37-40; and the method of Vanda disclosed by H. Y. Nakasone (1961), Technical Bulletin, Hawaii Agricultural Experimental Station, U.S.A. are successful in utilizing the chemical agent of Colchicine to treat with the protocorms or protocorm-like bodies (PLBs) for the production of polyploid plants. Although these chemical-induction techniques can be successful in chromosome doubling, they can have disadvantages of operating in complicated procedure and problems of dealing with the toxicity of chemical agents which are unacceptable.
Some papers known in the arts further demonstrate the occurrence of the phenomenon of endoreduplication in several orchid cultivars. For example, endoreduplication of Dendrobiutn is disclosed by W. E. Jones and A. R. Kuehnle (1998), Lindleyana, 13:11-18; the chromosome endoreduplication phenomenon of Phalaenopsis is disclosed by S. Lin et al. (2001), J. Amer. Soc. Hort. Sci., 126:195-199; the chromosome endoreduplication phenomenon of Spathoglottis is disclosed by M. Yang and C. S. Loh (2004), BMC Cell Biology, 5:33; and the chromosome endoreduplication phenomenon of Vanda is disclosed by M. Yang and C. S. Loh (2003), New phytologist, 159:659-667. The phenomenon of endoreduplication means that the DNA contained in the nucleus is replicated and no cell division occurs, which results in endopolyploidy. In Phalaenopsis orchid, the phenomenon of endoreduplication is found commonly in various plant tissues (e.g. protocom, leaf, root and corolla), which is disclosed by H. C. Lee et al. (2003), Plant Science, 166:549-667, for example.
On the other hand, some other papers known in the arts further demonstrate that culturing of the leaf or root of Phalaenopsis in a proper medium could produce PLBs to regenerate the plants as disclosed by Hsu and Chen (2003), Journal Chinese Society Horticultural Science, 49:335-342 and T. W. Yam and M. A. Weatherhead (1991), Lindleyana, 6:151-153.
Some other papers known in the arts yet further point out that a method of physical cutting on the protocorms or protocorm-like bodies (PLBs) could induce multiplication of PLBs. For example, the method of physical cutting on the protocorm of Phalaenopsis gigantea and culturing it in a coconut-water and activated-charcoal-contained medium could achieve high-frequency multiplication of Phalaenopsis gigantea seedlings, which is disclosed by R. Murdad et al. (2006), Scientia Horticulturae, 111:73-79.
JP6046700 discloses a method for PLB proliferation of Phalaenopsis . The method requires cutting a tip slice of PLBs and culturing in a solid or liquid medium to obtain regeneration of PLBs. The regenerated PLBs could be subcultured and further proliferated by using the same method.
Each of the above-mentioned papers and Japanese patent is incorporated herein by reference for purposes including, but are not limited to, indicating the background of the present invention and illustrating the state of the art.
As is described in above-referenced papers and patent, the present invention provides a new and effective method of producing polyploid plants of orchids for the use in hybridization or induced mutation breeding.